Oncogene (2012) 31, 4689 --4697 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc

ORIGINAL ARTICLE Regulation of IMP3 by EGFR signaling and repression by ERb: implications for triple-negative breast cancer

S Samanta1, VM Sharma1, A Khan2 and AM Mercurio1

Insulin-like growth factor II (IGF-II) mRNA-binding 3 (IMP3) is emerging as a useful indicator of the progression and outcome of several cancers. IMP3 expression is associated with triple-negative breast carcinomas (TNBCs), which are aggressive tumors associated with poor outcome. In this study, we addressed the hypothesis that signaling pathways, which are characteristic of TNBCs, impact the expression of IMP3 and that IMP3 contributes to the function of TNBCs. The data obtained reveal that IMP3 expression is repressed specifically by estrogen b (ERb) and its ligand 3bA-diol but not by ERa. Epidermal growth factor receptor (EGFR) signaling and consequent activation of the mitogen-activated protein kinase pathway induce IMP3 and expression. Interestingly, we discovered that the EGFR promoter contains an imperfect estrogen response element and that ERb represses EGFR transcription. These data support a mechanism in which ERb inhibits IMP3 expression indirectly by repressing the EGFR. This mechanism relates to the biology of TNBC, which is characterized by diminished ERb and increased EGFR expression. We also demonstrate that IMP3 contributes to the migration and invasion of breast carcinoma cells. Given that IMP3 is an mRNA-binding protein, we determined that it binds several key mRNAs that could contribute to migration and invasion, including CD164 (endolyn) and MMP9. Moreover, expression of these mRNAs is repressed by ERb and enhanced by EGFR signaling, consistent with our proposed mechanism for the regulation of IMP3 expression in breast cancer cells. Our findings show that IMP3 is an effector of EGFR-mediated migration and invasion and they provide the first indication of how this important mRNA-binding protein is regulated in cancer.

Oncogene (2012) 31, 4689--4697; doi:10.1038/onc.2011.620; published online 23 January 2012 Keywords: breast cancer; IMP3; ERb; EGFR

INTRODUCTION In this study, we addressed the hypothesis that signaling IMP3 is a member of a family of insulin-like growth factor II (IGF-II) pathways, which are characteristic of TNBCs, impact the expres- mRNA-binding (IMPs), consisting of IMP1, IMP2 and sion of IMP3 and that IMP3 contributes to the function of TNBCs. IMP3.1 IMPs have important roles in RNA trafficking, stabilization, A salient feature of TNBC is the absence of or diminished ER localization and migration, especially during early stages of signaling. More specifically, TNBCs are ERa negative and they 14 both human and mouse embryogenesis.2 IMPs are expressed in express low levels of ERb. A reasonable possibility based on developing , placenta and muscle, but they are these observations is that ER signaling represses IMP3. Interest- undetectable in normal adult tissues.3 Importantly, however, ingly, our data reveal that ERb and its ligand 3bA-diol (5a- IMP3 is re-expressed in several malignant tissues, including Androstane-3b,17b-diol) repress IMP3 expression but that ERa pancreatic, lung, renal cell, ovarian, endometrial and cervical does not contribute to this repression. Further analysis demon- cancers.4--9 This phenomenon has been exploited for the strated that epidermal growth factor receptor (EGFR) signaling prognostic assessment of specific cancers. In particular, IMP3 is induces IMP3 expression and that ERb represses EGFR transcrip- an accurate predictor of renal-cell carcinoma and tion, revealing a mechanism in which ERb inhibits IMP3 expression prognosis,4 and similar trends are emerging for other cancers.10 --12 indirectly by repressing the EGFR. This finding is relevant to the We are interested in breast cancer in this context, especially in biology of TNBC because these tumors are characterized by 15 of the finding that IMP3 is expressed preferentially in triple- overexpression of the EGFR and concomitant EGFR signaling. negative breast cancers.10 Triple-negative breast cancer (TNBC) is Our observation that IMP3 contributes to the migration and a molecular subtype of breast cancer characterized by the absence invasion of TNBC cells provides a functional role for this IMP in of estrogen receptor-a (ERa), progesterone receptor and human breast cancer, and it suggests that IMP3 can be an effector of epidermal growth factor receptor-2 amplification. Clinically, TNBCs EGFR-mediated migration and invasion. are usually of high histological grade, poorly differentiated and more aggressive compared with other subtypes of breast cancer.13 In fact, treatment of TNBC patients remains a challenge RESULTS because of the lack of targeted therapeutic options and the high ERb suppresses IMP3 expression metastatic potential of TNBC cells. These observations indicate To assess the possible role of ERs in regulating IMP3 expression, that IMP3 could prove useful for the clinical management of TNBC we used MDA-MB-231 and MDA-MB-468 cells, which are TNBC cell but much remains to be learned about its regulation and function lines that express IMP3 but lack ERa and express low levels of ERb. in these tumors. Exogenous expression of ERa in MDA-MB-231 cells did not alter

1Department of Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA and 2Department of Pathology, UMass Memorial Medical Center, Worcester, MA, USA. Correspondence: Dr AM Mercurio, Department of Cancer Biology, University of Massachusetts Medical School, 364 Plantation Street, Lazare Research Building, Worcester, MA 01605, USA. E-mail: [email protected] Received 25 September 2011; revised 17 November 2011; accepted 1 December 2011; published online 23 January 2012 Regulation of IMP3 in breast cancer S Samanta et al 4690 IMP3 expression (Figure 1a). Also, inhibition of endogenous ERa IMP3 expression significantly compared with IgG-treated cells activity by ICI-182780 (a selective ERa antagonist) was unable to (Figure 2a). This Ab also diminished EGFR phosphorylation and induce IMP3 expression in MCF7 cells, which express ERa and ERb MAP (mitogen-activated protein) kinase activation, which is a bona but not IMP3 (Figure 1a). Based on these results, we focused our fide downstream effector of the EGFR signaling pathway18 attention on ERb. PHTPP (4--2--phenyl-5, 7--bis (trifluoromethyl) (Figure 2a). To substantiate the involvement of EGFR signaling pyrazolo (1,5--a) pyrimidin-3--yl phenol) is a specific ERb pathway in regulating IMP3 expression, we inhibited MEK1/2 antagonist and it caused a significant increase in IMP3 expression (upstream component of MAPK) using two different inhibitors in both MDA-MB-231 and MDA-MB-468 cells (Figure 1b). This (PD98059 and U0126). As shown in Figure 2b, IMP3 mRNA and finding was substantiated using 3bA-diol, an androgen metabolite protein expression is reduced significantly upon treatment with that is a specific ligand of ERb.16 3bA-diol decreased IMP3 these inhibitors. Similar results were obtained using MDA-MB-468 expression in MDA-MB-231 cells indicating that ligand-dependent cells (Figure 2c). We also assayed the activity of the IMP3 promoter activation of ERb represses IMP3 (Figure 1c). Also, depletion of in the presence of the MEK1/2 inhibitors using a reporter construct ERb, using small hairpin RNAs, increased IMP3 expression in both containing the human IMP3 proximal promoter. As shown in MDA-MB-231 and MDA-MB-468 cells significantly (Figure 1d). Figure 2d, inhibition of MEK1/2 using either U0126 or PD98059 Interestingly, PHTPP did not induce IMP3 expression in MCF7 cells decreased luciferase activity considerably. Collectively, our data (Figure 1e), indicating that the ERb-mediated repression of IMP3 indicate that an EGFR/MEK/MAPK pathway regulates IMP3 may be restricted to TNBC cells. expression. We observed that estrogen (E2) increased IMP3 expression in MDA-MB-231 cells (Figure 1f). Although this result seems ERb suppresses EGFR expression unexpected, we also found that estrogen increased EGFR phosphorylation in these cells (Figure 1f) and that the ability of The contrasting data we obtained with ERb and EGFR regulation of estrogen to increase EGFR phosphorylation was not affected by IMP3 expression raised the possibility that these receptors function either inhibition (PHTPP) or stimulation (3bA-diol) of ERb function in a common pathway. This possibility was supported by our (Figure 1f). These findings are consistent with the report that observation that depletion of ERb expression in both MDA-MB-231 estrogen exerts its non-genomic action through EGFR signaling.17 and MDA-MB-468 cells increased EGFR mRNA and protein expres- sion significantly compared with control cells expressing GFP (green fluorescent protein) small hairpin RNA (Figures 3a and b). Similarly, EGFR signaling positively regulate IMP3 expression overexpression of ERb reduced EGFR protein and mRNA (Figure 3c). Given that high EGFR expression is associated with TNBC,15 we To substantiate our hypothesis, we inhibited ERb function using the assessed the possibility that EGFR signaling regulates IMP3 selective antagonist PHTPP and observed increased EGFR protein expression. Treatment of both MDA-MB-231 and MDA-MB- expression (Figure 3d). In contrast, PHTPP did not induce EGFR 468 cells with an EGFR function-blocking (Ab) reduced expression in MCF7 cells (Figure 3d). Interestingly, restoration of ERa

α ICI PHTPP PHTPP pcDNA3 pER Ctrl24 48 72 hr ERα MDA-231Ctrl 48 72 hr Ctrl 48 72 hr IMP3 IMP3 IMP3 IMP3 Actin Actin Actin Actin MDA-231 MCF7 MDA-231 MDA-468

1.5

# 1 # 2 β # 1 β # 2 β β 3β-adiol 1 Ctrl shRNAshER- shER- Ctrl shRNAshER- shER- Ctrl 48 72 hr ER-β ER-β IMP3 0.5 Fold change IMP3 * ** IMP3 Actin Actin Actin 0 MDA-231 MDA-468 Ctrl 48 hr 72 hr 3β-adiol

Estrogen βA-diol PHTPP Ctrl 2684 24 hr Ctrl E2 E2+PHTPPE2+3 Ctrl24 48 72 hr pEGFR MDA-231 IMP3 IMP3 IMP3 Actin Actin Actin MDA-231 MCF7 MDA-231 Figure 1. ERb suppresses IMP3 protein expression. (a, left) ERa was expressed in MDA-MB- 231 cells by transfecting them with an expression vector, pcDNA3/ERa (pERa). (a, right) MCF7 cells were treated with either dimethylsulphoxide or the ERa antagonist ICI (1 mM) and analyzed for IMP3 expression by immunoblotting. MDA-MB-231 cells were used as positive control for IMP3 expression. (b) and (e) Immunoblots show IMP3 expression in MDA-MB-231, MDA-MB-468 and MCF7 (e) cells treated with the ERb antagonist PHTPP (10 mM). (c) MDA-MB-231 cells were treated with 3bA-diol (10 mM). Graphs on the right side of blot represent densitometry analysis (Image J software, NIH, rsbweb.nih.gov/ij/) of IMP3 expression. (d) Blots show the effect of ERb depletion on IMP3 expression in MDA-MB-231 and MDA-MB-468 cells. ERb expression was depleted by transfecting cells with plasmids expressing two different small hairpin RNAs. A plasmid expressing green fluorescent protein- small hairpin RNA was used as control. (f, left) Immunoblot shows the effect of estrogen (E2,10nM) on IMP3 protein expression in MDA-MB- 231 cells. (f, right) Effect of PHTPP and 3bA-diol (24 h) on IMP3 expression in estrogen-treated MDA-MB-231 cells. The expression of IMP3, ERa and ERb was assessed 48 h post transfection. Dimethylsulphoxide was used as control for estrogen, PHTPP and 3bA-diol treatment.

Oncogene (2012) 4689 --4697 & 2012 Macmillan Publishers Limited Regulation of IMP3 in breast cancer S Samanta et al 4691 1.2 IgG Ab IgG Ab U0126 PD98059 0.9 pEGFR pEGFR Ctrl610 hr Ctrl610 hr ** 0.6 * pMAPK pMAPK pMAPK pMAPK 0.3 IMP3 IMP3 IMP3 IMP3 IMP3 mRNA (fold change) Actin Actin Actin Actin 0 Ctrl 6 hr 10 hr MDA-231 MDA-468 MDA-231 MDA-231 U0126

1.5 1.5 1.2 MDA-231 MDA-231 U0126 1 Ctrl610 hr 0.8 * 1 IMP3 * 0.4 0.5 0.5 pMAPK * IMP3 mRNA (fold change) Actin 0 RLU (Fold change) 0 RLU (Fold change) MDA-468 Ctrl U0126 0 Ctrl PD98059 Ctrl U0126 10 hr IMP3 promoter IMP3 promoter

Figure 2. EGFR signaling positively regulates IMP3 expression. (a) Immunoblots show the effect of blocking EGFR on IMP3 expression using a specific antibody (2 mg/ml) in MDA-MB-231 (left) and MDA-MB-468 (right) cells. Rabbit IgG was used as control. (b) and (c) MDA-MB-231 and MDA-MB-468 cells were treated with MEK1/2 inhibitors U0126 (10 mM) and PD98059 (50 mM) for different time points as indicated in the figure, and the expression of IMP3, as well as pMAPK, was analyzed by immunoblotting. IMP3 mRNA from cells treated with U0126 for 10 h was assessed by qPCR. (d) MDA-MB-231 cells were transfected with a luciferase reporter construct containing 2.872 kb IMP3 promoter (wild-type) in presence or absence of U0126 and PD98059, and luciferase activity was measured 24 h post transfection. Data represent the mean of three independent experiments. P-value (*) o0.05.

2.5 1.2 # 1 # 2 β # 1 β # 2 β β β 2 * ** pcDNA3pER 0.9 shER- shER- Ctrl shRNAshER- shER- 1.5 Ctrl shRNA β ERβ 0.6 * ERβ 1 ER- EGFR EGFR 0.5 EGFR 0.3 EGFR mRNA (fold change) EGFR mRNA (fold change) Actin 0 Actin Actin 0 MDA-468 MDA-231 β MDA-231 # 1 # 2 β β pER pcDNA3 Ctrl shRNAshER shER

PHTPP PHTPP PHTPP α Ctrl 24 48 72 hr Ctrl 48 72 hr Ctrl24 48 72 hr MDA-231 pcDNA3 pER α EGFR EGFR EGFR ER EGFR Actin Actin Actin Actin MDA-231 MDA-468 MCF7

3

EGFR promoter * + 212 bp 2 -1.984 kb ERE RLU AATCAACGTGAAT TSS 1 (Fold change)

Mutated ERE: AACATACGTGAAT 0

Mutant Wild type

Figure 3. ERb suppresses EGFR expression. (a) and (b)ERb was transiently depleted in MDA-MB-231 and MDA-MB-468 cells using two different small hairpin RNAs. EGFR protein and mRNA (MDA-MB-231) expression was evaluated by immunoblotting and qPCR, respectively. (c)ERb was overexpressed in MDA-MB-231 cells using an expression construct (pERb), and EGFR protein and mRNA expression was evaluated by immunoblotting and qPCR, respectively. (d) MDA-MB-231, MDA-MB-468 and MCF7 cells were treated with the ERb antagonist PHTPP (10 mM) and EGFR protein expression was analyzed by immunoblotting. (e)ERa was expressed in MDA-MB-231 cells using an expression construct (pERa) and EGFR protein was assessed by immunoblotting. (f) Luciferase reporter constructs containing the wild-type 2.2-kb EGFR promoter and the mutant (mutated ERE) were transfected into MDA-MB-231 cells and luciferase activity was measured 24 h post-transfection. Data represent the mean of three independent experiments. The empty vector pcDNA3 was used as control. P-value (*) o0.05.

& 2012 Macmillan Publishers Limited Oncogene (2012) 4689 --4697 Regulation of IMP3 in breast cancer S Samanta et al 4692 expression in MDA-MB-231 cells did not affect EGFR expression constructs revealed that mutation of the imperfect ERE increased (Figure 3e), consistent with our finding that ERa does not contribute luciferase activity by B2-fold suggesting that this sequence has a to the regulation of IMP3. repressive role in EGFR transcription (Figure 3f). The EGFR promoter contains an imperfect estrogen response element (ERE) at 1.949 kb (AATCAACGTGAAT) suggestive of the possibility that ERb may repress EGFR transcription. This imperfect IMP3 increases migration and invasion of breast cancer cells ERE was identified on the basis of its homology with the imperfect Given that triple-negative breast cancers are more aggressive, ERE present in the human vascular endothelial growth factor possess high metastatic potential and overexpress IMP3,10,13 it is promoter, which is 50-AATCAGACTGACT-30.19 The putative ERE reasonable to hypothesize that IMP3 contributes to their present in the EGFR promoter differs from that in the vascular aggressive behavior. To address this hypothesis, we depleted endothelial growth factor promoter by a single nucleotide ‘A’ as IMP3 expression in MDA-MB-231 cells using siRNA and assessed shown in italics, whereas the imperfect ERE present in the vascular their ability to migrate and invade using transwell assays. As endothelial growth factor promoter differs from the perfect ERE shown in Figure 4a, depletion of IMP3 reduced migration and (GGTCAnnnTGACC)20 by three nucleotides (underlined). This invasion significantly. Moreover, expression of IMP3 in MCF7 cells, observation suggested that the core ERE sequence TCAnnnTGA a non-triple-negative and non-invasive cell line increased their is critical for the sequence to be functional. To assess the migration and invasion (Figures 4b and c). A functional link contribution of this sequence to EGFR regulation, we generated between ERb and IMP3 is supported by our finding that ERb reporter constructs containing the 2.2-kb wild-type EGFR promo- expression represses migration and invasion as evidenced by ter or this construct containing a mutation in the core sequence modulation of its expression in MDA-MB-231 cells (Figures 4d (TCAACGTGA to CATACGTGA). The data obtained with these and e). Moreover, a functional link between EGFR signaling and

a 150 Migration 150 Invasion

100 100 Ctrlsi siIMP3 * * IMP3 50 50 (% control) Actin (% control) No. of cells/field No. of cells/field MDA- 231 0 0 CtrlsisiIMP3 Ctrlsi siIMP3

Migration Invasion b c 200 200 8000 * * 150 150 -231 A D 4000 100 100 M pcDNA4pIMP3 (% control) IMP3 (% control) 50 50 IMP3 mRNA (fold change) No. of cells/field No. of cells/field Actin 0 0 0 MCF7 pIMP3 pIMP3 pIMP3 pcDNA4 pcDNA4 pcDNA4

de300 Migration 300 Invasion150 Migration 150 Invasion ** * * 200 ** 200 100 100 * * 100 100 50 50 (% control) (% control) (% control) (% control) No. of cells/field No. of cells/field No. of cells/field No. of cells/field 0 0 0 0 β β β # 1 β # 2 β # 1 β # 2 pER pER pcDNA3 pcDNA3 Ctrl shRNAshER shER Ctrl shRNAshER shER

f Migration Invasion 120 120

90 90 * * 60 60 (% control) 30 (% control) 30 No. of cells/field No. of cells/field 0 0

DMSO U0126 DMSO U0126 Figure 4. IMP3 enhances the migration and invasion of breast cancer cells. (a) IMP3 was transiently depleted in MDA-MB-231 cells using siRNA (20 nM). At 72 h post-transfection, IMP3 protein was evaluated by immunoblotting, and migration and invasion assays were performed for another 4 h. (b) and (c) IMP3 was expressed transiently in MCF7 cells by transfecting with an expression construct (pIMP3) and IMP3 protein and mRNA were determined 48 h post transfection by immunoblotting and qPCR, respectively. The same IMP3-expressing cells were subjected to migration and invasion assays for another 24 h. The empty vector pcDNA4/His/Max was used as control. (d) and (e) Migration and invasion assays were carried out with ERb-depleted cells, as well as cells overexpressing ERb.(f) MDA-MB-231 cells were treated with the MEK 1/2 inhibitor U0126 and assayed for migration and invasion. Data are representative of three independent experiments. P-value (*) o0.05.

Oncogene (2012) 4689 --4697 & 2012 Macmillan Publishers Limited Regulation of IMP3 in breast cancer S Samanta et al 4693 IMP3 is supported by the observation that U0126, a MEK inhibitor, Based on our findings, IMP3 mRNA targets that we identified reduced migration and invasion significantly (Figure 4f). should be regulated by ERb and EGFR. Indeed, depletion of ERb The fact that IMP3 is an mRNA-binding protein suggests that it increased the expression of MMP9 and CD164 mRNAs significantly contributes to migration and invasion by regulating specific in MDA-MB-231 cells (Figure 6a). In contrast, Ab-mediated mRNAs. The obvious target for IMP3 is IGF-II but depletion of IMP3 inhibition of the EGFR decreased the expression of these mRNAs in MDA-MB-231 cells or expression in MCF-7 cells had no effect on (Figure 6b). We also observed a significant reduction in the IGF-II mRNA expression (Figures 5a and b). Several putative mRNA expression of these mRNAs in cells treated with MEK1/2 inhibitor targets of IMP3 were recently identified based upon PAR-CLIP U0126 (Figure 6c). (photoactivatable-ribonucleoside-enhanced-crosslinking and im- munoprecipitation)21 that could contribute to cell motility and invasion. These targets include CD164 (endolyn), RhoA, CCNG1 and DISCUSSION CCND2, which are known to have a role in carcinogenesis.22 --25 We The data obtained in this study provide insight into the assayed these candidate mRNAs by qPCR (quantitative PCR) in mechanisms that regulate the expression of IMP3 in breast IMP3-depleted MDA-MB-231 cells and in MCF7 cells engineered to carcinoma cells and that are associated with the function of this express IMP3. Although no significant change in RhoA, CCNG1 and mRNA-binding protein in migration and invasion. Our data CCND2 mRNA expression was observed (data not shown), CD164 revealed that IMP3 expression is repressed by ERb but not ERa, mRNA expression was significantly decreased in IMP3-depleted and that it is stimulated by EGFR signaling. More specifically, we MDA-MB-231 cells and it was increased in IMP3-expressing MCF7 demonstrate that ERb represses transcription of the EGFR and that cells (Figures 5c and d). We also assayed the hyaluronan receptor EGFR signaling regulates IMP3 expression directly (Figure 7). An CD44 expression because it is known to contribute to tumor important and novel implication of this study is that IMP3 is an progression and there is evidence that it can be regulated by IMP3 effector of EGFR-driven migration and invasion. Our findings have in HeLa cells.26 Our data, however, did not support IMP3-mediated important implications for the biology and therapy of TNBC, which regulation of CD44 mRNA expression in breast carcinoma cells is characterized by elevated expression of EGFR and IMP3,10,28 and (Figure 5e). CD44 functions as a docking site for the matrix reduced expression of ERb.14 metalloproteinase MMP9, which contributes to invasion.27 Our results highlight a role for ERb in regulating the functions of Although MMP9 had not been identified as an IMP3 target, breast carcinoma cells that is distinct from ERa. It is surprising that however, we observed that MMP9 mRNA expression is regulated a role for ERa in regulating IMP3 expression was not revealed by by IMP3 (Figures 5c and d). We performed Ribo-immunoprecipita- our data because loss of ERa is a defining characteristic of TNBC.29 tion-qPCR analysis to establish that IMP3 indeed binds with IGF-II, Establishing a distinct function for ERb is significant because much MMP9 and CD164 mRNA (Figure 5f). less is known about ERb than ERa in breast cancer biology.

1.5 1.5

1 1 Ctrlsi siIMP3 MDA-231pcDNA4pIMP3 0.5 IMP3 0.5 IMP3 IGF2 mRNA (fold change) IGF2 mRNA Actin (fold change) Actin 0 0 MDA-231 Ctrlsi siIMP3 MCF7 pIMP3 MDA-231 pcDNA4

1.5 ctrlsi siIMP3 5 pcDNA4 pIMP3 1.2 4 Ctrlsi siIMP3 0.9 3 * * IMP3 0.6 2 Actin 0.3 1 * * mRNA fold change

MDA-231 mRNA fold change 0 0 CD164 MMP9 CD164 MMP9 MDA-231 MCF7 f 1.5 3 IgG2a IMP3 * * * 1 2

Ctrlsi siIMP3 0.5 1 CD44 CD44 mRNA (fold change)

Actin mRNA fold change 0 0 MDA-231 Ctrlsi siIMP3 CD164 MMP9 IGF2 MDA-231 MDA-231: RIP-qPCR Figure 5. IMP3 binds and stabilizes CD164 and MMP9 mRNA. (a) and (b) IGF-II mRNA expression was quantified using qPCR in IMP3-depleted MDA-MB-231 cells and in MCF7 cells engineered to express IMP3. The blot in (a) shows IMP3 depletion in MDA-MB-231 cells and in (b) IMP3 expression in MCF-7 cells. (c) and (d) CD164 and MMP9 mRNA expression was quantified using qPCR in IMP3-depleted MDA-MB-231 cells and MCF-7 cells expressing IMP3. (e) CD44 protein and mRNA expression was assessed in IMP3-depleted MDA-MB-231 cells. (f) IMP3-associated RNAs in cytoplasmic extracts of MDA-MB-231 cells were isolated using an IMP3 antibody (25 mg). Non-immune rabbit IgG was used as negative control. Expression of CD164, MMP9 and IGF-II mRNAs was quantified using qPCR. Data are representative of three independent experiments. P-value (*) o0.05.

& 2012 Macmillan Publishers Limited Oncogene (2012) 4689 --4697 Regulation of IMP3 in breast cancer S Samanta et al 4694 abc MMP9 4 CD164 1.5 5 ** 1.5 IgG Ab DMSO ** U0126 4 3 * 1 1 3 2 * 2 * 0.5 0.5 * * 1 1 * mRNA fold change mRNA fold change 0 0 mRNA fold change 0 mRNA fold change 0 MMP9 CD164 MMP9 CD164 # 1 # 2 β # 1 β # 2 β β

Ctrl shRNAshER shER Ctrl shRNAshER shER Figure 6. CD164 and MMP9 mRNAs are suppressed by ERb and increased by EGFR signaling. (a-- c) Expression of CD164 and MMP9 mRNAs was quantified by qPCR in MDA-MB-231 cells in which ERb had been depleted or overexpressed, or had been treated with either an EGFR antibody or the MEK 1/2 inhibitor U0126. Each experiment was repeated at least three times. P-value (*), (**)o0.05.

15 Model figure survival and invasion of tumor cells. Enhanced expression of CD164 EGFR in primary breast tumors predicts poor prognosis and 34 --37 3βA-diol mRNA renders them more aggressive. In the context of our findings, EGFR-driven migration and invasion contribute to tumor progres- Migration & sion and metastasis, and the mechanisms involved have been the EGFR IMP3 38 --40 ERβ invasion focus of numerous studies. We conclude from our findings MMP9 that IMP3 can be an effector of EGFR-driven migration and mRNA invasion, and suggest that this contribution of IMP3 needs to be integrated with other mechanisms of EGFR-mediated migration Figure 7. Proposed model for how ERb regulates IMP3 in triple- and invasion. From a clinical perspective, the EGFR is considered negative breast cancer cells. The interaction of ERb with its ligand to be a viable target for therapy of TNBC. Although small-molecule 3bA-diol suppresses EGFR expression. EGFR signaling induces IMP3 EGFR inhibitors, including gefitinib (Iressa) and the monoclonal expression. IMP3 facilitates the migration and invasion of triple- antibody cetuximab, are in clinical trials for this aggressive negative breast carcinoma cells by a mechanism that may involve its subtype of breast cancer,41 none of these drugs has shown ability to bind specific mRNAs, such as CD164 and MMP9. efficacy till date. We postulate based on our data that small- molecule inhibitors of IMP3 would augment anti-EGFR therapy for the treatment of TNBC. In addition to its ability to repress IMP3, ERb has been shown to The overarching issue raised by our data is how IMP3 inhibit breast proliferation and invasion by suppressing contributes to the aggressive behavior of breast cancers. In fact, cell cycle , including c-Myc, cyclin D1 and cyclin A.30 The this issue is important for several cancers in which IMP3 report that ERb expression correlates inversely with tumor grade14 expression has been correlated with progression and poor supports our conclusion that ERb represses IMP3 because TNBC is outcome.4,7,10 A role for IMP3 in invasion was foreshadowed by usually high grade. Our data also indicate that there is ligand the finding that IMPs (specifically IMP1 and IMP3) induce specificity in the regulation of IMP3 expression. Specifically, we invadopodia formation by regulating the expression of CD44 in demonstrated that 3bA-diol functions as an ERb-specific ligand HeLa cells.26 Although our data did not reveal that CD44 that represses IMP3 expression. Estrogen, in contrast, actually expression is regulated by IMP3 in breast cancer, the induction increased IMP3 expression in TNBC cells. Given that these cells are of invadopodia formation by IMPs is consistent with our data. ERa-negative, we conclude that this effect of estrogen is non- Recent studies have implicated IMP3 in the migration of HeLa genomic and it may be explained by its effect on EGFR signaling.17 cells6 and in the aggressive behavior of glioblastoma cells This conclusion is supported by our observation that estrogen including invasion.42 In addition to a role in migration and increased EGFR phosphorylation in ERa-negative cells indepen- invasion, IMP3 has been implicated in proliferation, anchorage- dently of ERb function. dependent and survival.42,43 However, depletion of An unexpected and novel finding in this study is that ERb IMP3 expression in breast carcinoma cells did not affect cell cycle represses EGFR expression and signaling, possibly through its and anchorage-dependent growth (data not shown). ability to repress EGFR transcription. Our identification of an Given that IMP3 is an mRNA-binding protein, the most feasible imperfect ERE sequence in the EGFR promoter and demonstration hypothesis to account for its role in migration and invasion is that that this sequence represses EGFR transcription supports this it affects the expression or localization of specific mRNAs that are hypothesis. Moreover, there is precedence in the literature that critical for invasion and progression. Our finding that IMP3 binds ERb can repress transcription by recruiting co-repressors such as and stabilizes CD164 and MMP9 mRNAs provides potential insight NCoR.31 There is also indirect evidence that expression of ERb in into this hypothesis. CD164, a cell surface receptor for sialomucin, malignant pleural mesothelioma (MME) cells impedes EGFR has been implicated in metastasis22 and MMP9, a signaling.32 An important issue that arises from these observations type-IV collagenase, contributes to carcinogenesis by several is the extent to which diminished ERb expression in TNBC mechanisms including degradation of the basement membrane44 contributes to the elevated expression of EGFR that is character- and promoting the release of growth factors from the extracellular istic of this breast cancer subtype. There is evidence that EGFR matrix.45 Moreover, MMP9 expression is correlated with poor amplification is more common in TNBCs than non-TNBCs,33 prognosis in node-negative breast cancer.46 Another relevant but the possibility that a reduction in ERb expression functions in observation in our study is that IMP3 binds IGF-II and CD44 mRNAs concert with gene amplification should be considered. but it does not alter their expression. This finding is distinct from The relationship between EGFR and IMP3 that we uncovered other reports that have shown that IMP3 can activate the has important implications for the biology and therapy of breast translation of IGF-II in leukemia cells 47 and glioblastoma cells,42 cancer. EGFR signaling has profound effects on the proliferation, and regulate the expression of CD44 in Hela cells.26 The possibility

Oncogene (2012) 4689 --4697 & 2012 Macmillan Publishers Limited Regulation of IMP3 in breast cancer S Samanta et al 4695 that IMP3 regulates the localization or other aspects of these EGFR-forward: 50-GCCCCCACTGCGTCAAGACC-30 mRNAs merits investigation because both IGF-II and CD44 are EGFR-reverse: 50-ACCTGGCCCAGTGCATCCGT-30 important in the biology of breast cancer.48,49 ERb-forward: 50-AAGGTTAGTGGGAACCGTTG-30 In summary, our data provide the first insight into mechanisms ERb-reverse: 50-ACATCCTTCACACGACCAGA-30 that regulate the expression of an mRNA-binding protein that is MMP9-forward: 50-TTTGACAGCGACAAGAAGTGG-30 associated with TNBC and that is prognostic for progression and MMP9-reverse: 50-AGGGCGAGGACCATAGAGG-30 outcome in other cancers. These regulatory mechanisms are CD164-forward: 50-GAGTGCTGTAGGATTAATTGGAAAAT-30 intimately linked to the biology of TNBC and they also reveal a CD164-reverse: 50-GGGAGGAATGGAATTCTGC-30 novel mechanism of EGFR-driven migration and invasion. Our data IGFII-forward: 50-CCGAAACAGGCTACTCTCCT-30 also suggest that targeted-IMP3 therapy could augment EGFR IGFII-reverse: 50-AGGGTGTTTAAAGCCAATCG-30 inhibition in the treatment of TNBC. CD44-forward: 50-GCCCTTCCATAGCCTAATCC-30 CD44-reverse: 50-CTTTGGTGTCTCCCAGAAGC-30.

MATERIALS AND METHODS Cells, reagents and treatments Migration and invasion assays The breast cancer cell lines MDA-MB-231, MDA-MB-468 (mentioned as Assays were performed using transwell chambers (8-mm polycarbonate MDA-231 and MDA-468 in the figures) and MCF7 were obtained from membrane, Costar, Corning Inc, Horseheads, NY, USA) that had been American Type Culture Collection and maintained in Dulbecco’s modified coated with either I or Matrigel (BD Biosciences, Billerica, MD, 51 Eagle medium (low glucose) containing 10% fetal bovine serum, 1% USA) for migration and invasion, respectively, as described previously. streptomycin and penicillin at 37 1C in an incubator supplied with 5% CO2. Assays were quantified by counting the number of stained nuclei in five IMP3-specific siRNA was purchased from Santa Cruz Biotechnology, Inc. independent fields in each transwell. (Santa Cruz, CA, USA). A non-targeting pool was used as negative control. Dharmafect-4 was used for siRNA delivery and was purchased from Thermo Scientific (Rockford, IL, USA). Lipofectamine-2000 was used for Ribo-immunoprecipitation-qPCR regular DNA transfection and purchased from Invitrogen (Grand Island, NY, Identification of endogenous mRNA targets of IMP3 was performed by USA). The control plasmid vector pcDNA3 was purchased from Invitrogen riboimmunoprecipitation assay, as described previously with minor 47 7 and pcDNA4/His/Max was a gift from Dr Michael Green, UMass Medical modifications. Briefly, MDA-MB-231 cells (B2 Â 10 ) were harvested School. The lentiviruses (PLKO.1) containing ER-b small hairpin RNAs or and extracted for 15 min on ice in 250 ml of ice-cold lysis buffer (100 mm shGFP (Open Biosystems, Lafayette, CO, USA, clone ID: TRCN0000003326, KCl, 5 mm MgCl2, 10 mm HEPES (pH 7.0), 0.5% Nonidet P-40, 10 mm TRCN0000003327 and RHS4459) were generated in HEK293T cells and dithiothreitol) supplemented with RNase and inhibitors (1 ml of infected in MDA-MB-231 cells following standard protocols described lysis buffer contains 5.25 ml of 40 units/ml RNase OUT (Invitrogen), 2 mlof elsewhere.50 The MEK1/2 inhibitors U0126 and PD98059 were purchased 0.2% vanadyl ribonucleoside complexes (New England Biolabs, Beverly, from LC Laboratories (Woburn, MA, USA). ICI was purchased from Sigma- MA, USA), 50 ml of complete protease inhibitor mixture (Roche Applied Aldrich (St Louis, MO, USA). The IMP3 antibody was obtained from DAKO, Science). Extracts were cleared by centrifugation for 15 min at 13 000 r.p.m. (Hamburg, Germany). ERa, EGFR and pEGFR (Tyr-1263) Abs were purchased and supernatant was transferred to a fresh 1.5-ml tube. To pre-clear the from Santa Cruz Biotechnology Inc. pMAPK (p42/44) and pAkt (ser473) Abs cytoplasmic extracts, 25 mg of non-immune rabbit IgG (Sigma-Aldrich) was were obtained from Cell Signaling Technology (Danvers, MA, USA). The ERb added to the supernatant and kept on ice for 45 min, then incubated with antibody was purchased from Gen Tex Inc (Irvine, CA, USA). The IGF-II and 50 ml of a 50% (v/v) suspension of protein G-Sepharose beads (Biovision, CD44 were purchased from Abcam (Cambridge, MA, USA). San Francisco, CA, USA) for 3 h at 4 1C with rotation. This was centrifuged at

Estrogen (E2), PHTPP and 3bA-diol were purchased from Tocris Biosciences 13 000 r.p.m. and the supernatant was recovered (pre-cleared lysate). For (Minneapolis, MN, USA) and dissolved in dimethylsulphoxideDMSO. For immunoprecipitation, the pre-cleared extract was incubated with 100 mlof 3bA-diol treatment, MDA-MB-231 cells were cultured in DMEM (low a 50% suspension of protein G-Sepharose beads (Sigma-Aldrich) pre- glucose) supplemented with 5% charcoal-stripped fetal bovine serum. For coated with the same amount of either non-immune rabbit IgG (Sigma- treatment with all other reagents, cells were cultured in normal medium Aldrich) or anti-human IMP3 antibody (25 mg) in 800 ml of NT-2 buffer (mentioned above) and serum-starved for 24 h prior to treatment. (150 mm NaCl, 1 mm MgCl2, 50 mm Tris--HCl (pH 7.4), 0.05% Nonidet P-40) containing RNase inhibitor and protease inhibitors for overnight at 4 1C Immunoblotting with rotation. Beads were washed 10 times using ice-cold NT-2 buffer, digested with 20 units of RNase-free DNase I (Promega, Madison, WI, USA) Whole cell extracts were prepared using radioimmunoprecipitation assay in 100 ml of NT-2 buffer for 20 min at 30 1C, washed with NT-2 buffer, and buffer containing EDTA and ethylene glycol tetraacetic acid (Boston further digested with 0.5 mg/ml protease K (Ambion, Grand Island, NY, Bioproducts, Ashland, MA, USA). A protease- and phosphatase-inhibitor USA) in 100 ml of NT-2 buffer containing 0.1% sodium dodecyl sulphate at cocktail was added separately (Roche Applied Science, Indianapolis, IN, 55 1C for 30 min. RNA was extracted with Trizol (Invitrogen). Glycogen USA). Extracts (40--50 mg protein) were blotted with the appropriate (Roche Applied Science) was added to facilitate precipitation of RNA. Real- primary Abs and then incubated with mouse or rabbit IgG horseradish time PCR was performed with equal amount of RNA to detect IGF-II, MMP9 peroxidase-conjugated secondary antibody. ECL (Pierce Biotechnology, and CD164 mRNAs using previously described primers. Rockford, IL, USA) was used to develop the blots.

RNA isolation and real-time PCR analysis IMP3 and EGFR promoter analysis Total RNA was isolated from cultured cells using Trizol reagent (Invitrogen) The human IMP3 promoter (2.872 kb) was PCR amplified from human following the manufacturer’s protocol. Complementary DNA was synthe- genomic DNA. The PCR-amplified fragments were confirmed by restriction sized using superscript-II reverse transcriptase (Invitrogen). mRNAs were mapping and cloned at the NheI-Hind III sites into the pGL3 basic vector quantified by real-time PCR analysis (ABI Prism, Applied Biosystems, (Promega). Similarly, the EGFR promoter (2.2 kb) was cloned and ERE Carlsbad, CA, USA) using Power Syber Green PCR master mix (Applied (AATCAACGTGAAT) was mutated to (AACATACGTGAAT) using site-directed Biosystems). Quantification was performed using DDCt method and mutagenesis (QuikChange XL site-directed mutagenesis kit, Agilent GAPDH was used as reference gene. The following primer pairs were Technologies, Santa Clara, CA, USA). Primer pairs used: used for real-time PCR analysis: IMP3 promoter: Forward: 50-TATGATGCTAGCACTTGAAGTGCTAGTGCA IMP3-forward: 50-CCGCAGTTTGAGCAATCAGAA-30 AGACAACT IMP3-reverse: 50-CGAGAAAGCTGCTTGATGTGC-30 Reverse: 50-CAGTAGAAGCTTTCCACCAGTCTTCCTAAGTCTTAGGA

& 2012 Macmillan Publishers Limited Oncogene (2012) 4689 --4697 Regulation of IMP3 in breast cancer S Samanta et al 4696 0 EGFR promoter: Forward: 5 -TAGAAGACGCGTCAGGTACTAGCCAAGGA 9 Zheng W, Yi X, Fadare O, Liang SX, Martel M, Schwartz PE et al. The oncofetal CTACA protein IMP3: a novel biomarker for endometrial serous carcinoma. Am J Surg Reverse: 50-AATCGTCTCGAGGATCAATACTGGACGGAGTCAG Pathol 2008; 32: 304 --315. 10 Walter O, Prasad M, Lu S, Quinlan RM, Edmiston KL, Khan A. IMP3 is a novel biomarker for triple negative invasive mammary carcinoma associated with a Luciferase reporter assays more aggressive phenotype. Hum Pathol 2009; 40: 1528 --1533. MDA-MB-231 cells were transfected with the desired luciferase reporter 11 Li S, Cha J, Kim J, Kim KY, Kim HJ, Nam W et al. Insulin-like growth factor II mRNA- constructs (express firefly luciferase) along with another construct binding protein 3: a novel prognostic biomarker for oral squamous cell (expresses renilla luciferase) to normalize the transfection efficiency. carcinoma. Head Neck 2011; 33: 368 --374. Relative light unit was calculated as the ratio of firefly luciferase to renilla 12 Li D, Yan D, Tang H, Zhou C, Fan J, Li S et al. IMP3 is a novel prognostic marker luciferase activity (normalized luciferase activity). The protocol used for that correlates with colon cancer progression and pathogenesis. Ann Surg Oncol 2009; 16: 3499 --3506. transfection and measurement of luciferase activity has been described 52 13 Irvin Jr WJ, Carey LA. What is triple-negative breast cancer? Eur J Cancer 2008; 44: previously. Luciferase activity was measured in Beckman Coultier, DTX 2799 --2805. 880 luminomiter (Danvers, MA, USA). 14 Marotti JD, Collins LC, Hu R, Tamimi RM. 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